U.S. patent number 9,080,203 [Application Number 14/475,617] was granted by the patent office on 2015-07-14 for method and system for automated image analysis in cancer cells.
This patent grant is currently assigned to NeoDiagnostix, Inc.. The grantee listed for this patent is NeoDiagnostix Inc.. Invention is credited to Colyn Cain, Gregory Anton Endress, Elizabeth Light, Madhvi Upender.
United States Patent |
9,080,203 |
Endress , et al. |
July 14, 2015 |
Method and system for automated image analysis in cancer cells
Abstract
A method of screening for the presence and/or extent of a
pathology in a subject, the pathology characterized by an abnormal
chromosomal component in a cell of the subject, comprising the
steps of: contacting a biological sample comprising cell nuclei
from said subject with, one or more distinguishable labeled probes
directed to at least one chromosomal sequence that characterizes
the abnormality under conditions that promote hybridization of the
one or more probes to the at least one sequence, automatically
obtaining a representation of the one or more distinguishable
labels hybridized to the chromosomal sequences, automatically
analyzing the distribution and intensity of binding of the one or
more labels in the representation to determine the presence and/or
extent of an abnormal chromosomal component; and automatically
reporting results of the analysis; wherein the steps are carried
out without intervention by a human.
Inventors: |
Endress; Gregory Anton
(Belchertown, MA), Upender; Madhvi (Potomac, MD), Light;
Elizabeth (Gaithersburg, MD), Cain; Colyn (Bethesda,
MD) |
Applicant: |
Name |
City |
State |
Country |
Type |
NeoDiagnostix Inc. |
Gaithersburg |
MD |
US |
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Assignee: |
NeoDiagnostix, Inc. (Rockville,
MD)
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Family
ID: |
43499647 |
Appl.
No.: |
14/475,617 |
Filed: |
September 3, 2014 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20150024395 A1 |
Jan 22, 2015 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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14069432 |
Nov 1, 2013 |
8852865 |
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12840927 |
Dec 10, 2013 |
8603747 |
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61227270 |
Jul 21, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q
1/6827 (20130101); C12Q 1/6841 (20130101); G06T
7/0012 (20130101); G06K 9/00147 (20130101); C12Q
1/6816 (20130101); C12Q 1/6841 (20130101); C12Q
2565/601 (20130101); G06T 2207/10024 (20130101); G06T
2207/10056 (20130101); G06T 2207/30024 (20130101); G06T
2207/10064 (20130101) |
Current International
Class: |
C12Q
1/68 (20060101); G06T 7/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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Jan 2005 |
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WO |
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WO 2005001137 |
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Jan 2005 |
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WO |
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WO2004058050 |
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Feb 2005 |
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WO |
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WO2006002378 |
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Jul 2007 |
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WO |
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2008070333 |
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Jun 2008 |
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WO |
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WO2008070333 |
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Dec 2008 |
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WO |
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WO2010011683 |
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Apr 2010 |
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WO |
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2011011527 |
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May 2011 |
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WO |
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Other References
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Primary Examiner: Joike; Michele K
Assistant Examiner: Brown; Mindy G
Attorney, Agent or Firm: Wales; Michele M. InHouse Patent
Counsel, LLC
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuing application of and claims priority
to U.S. Ser. No. 14/069,432 which was filed on Nov. 1, 2013, which
is a continuing application of and claims priority to U.S. Ser. No.
12/840,927 which was filed on Jul. 21, 2010, now issued as U.S.
Pat. No. 8,603,747, which is a non-provisional of and claims
priority to U.S. Ser. No. 61/227,270 which was filed on Jul. 21,
2009. The entire contents of Ser. Nos. 14/069,432, 12/840,927 and
61/227,270 are hereby incorporated by reference in their entirety.
Claims
What is claimed is:
1. An automated method for detecting chromosomal abnormalities in a
plurality of cells in a cervical sample, said method comprising: a)
hybridizing nucleic acid probes to a target, wherein the target
comprises EVII or MDS1; b) detecting the hybridization signal of
the nucleic acid probes, wherein the hybridization signal is
indicative of chromosomal copy number for the target; c) scoring
the chromosomal copy number of the target ; and d) reporting
whether the sample contains chromosomal abnormalities wherein the
sample is determined to be negative or have normal ploidy when a
gain of chromosomal copy number is in less than 0.9% of the
cells.
2. The method of claim 1, wherein the scoring of the chromosomal
copy number is performed by counting the cells having chromosomal
abnormalities.
3. The method of claim 1, wherein the scoring of the chromosomal
copy number is performed by counting hybridization signals.
4. The method according to claim 1, wherein the method is performed
by using an automated microscope.
5. The method according to claim 1, wherein the cells in said
sample are deposited in a thin layer on a slide.
6. The method of claim 1, wherein the hybridization is detected by
FISH, CISH, PCR, ELISA, CGH, Array CGH or flow cytometry.
7. The method of claim 1, wherein the target further comprises
TERC, APRM1, or MYNN.
8. The method of claim 7, wherein the target further comprises a
locus at 5p15, 5p15.3, or 5p15.2.
9. The method of claim 7, wherein the target further comprises
TERT, TRIP13, or Cri du Chat locus at 5p15.2.
10. The method of claim 7, wherein the target further comprises
CEN7 or CEN3.
11. The method of claim 7, wherein the target further comprises a
locus at chromosome 1q or 20q.
12. The method of claim 7, wherein at least 800 cells are
examined.
13. The method of claim 7, wherein at least 1000 cells are
examined.
14. The method of claim 7, wherein the target comprises TERC,
APRM1, and MYNN.
15. The method of claim 7, wherein the target further comprises
LRRC34.
16. The method of claim 15, wherein the target further comprises a
locus at 5p15, 5p15.3, or 5p15.2.
17. The method of claim 15, wherein the target further comprises
TERT, TRIP13, or Cri du Chat locus at 5p15.2.
18. The method of claim 15, wherein the target further comprises
CEN7 or CEN3.
19. The method of claim 15, wherein the target further comprises a
locus at chromosome 1q or 20q.
20. The method of claim 15, wherein at least 800 cells are
examined.
21. The method of claim 15, wherein at least 1000 cells are
examined.
22. The method of claim 15, wherein the target further comprises
SAMD7, LOC1000128164, SEC62, GPR160, PHC3, GPR160, PHC3 or
PRKCI.
23. The method of claim 22, wherein the target further comprises a
locus at 5p15, 5p15.3, or 5p15.2.
24. The method of claim 22, wherein the target further comprises
TERT, TRIP13, or Cri du Chat locus at 5p15.2.
25. The method of claim 22, wherein the target further comprises
CEN7 or CEN3.
26. The method of claim 22, wherein the target further comprises a
locus at chromosome 1q or 20q.
27. The method of claim 22, wherein at least 800 cells are
examined.
28. The method of claim 22, wherein at least 1000 cells are
examined.
29. The method of claim 22, wherein the target comprises SAMD7,
LOC1000128164, SEC62, GPR160, and PHC3.
30. The method of claim 22, wherein the target comprises GPR160,
PHC3, and PRKCI.
31. The method of claim 7, wherein at least 50 cells are
examined.
32. The method of claim 15, wherein at least 50 cells are
examined.
33. The method of claim 22, wherein at least 50 cells are examined.
Description
FIELD
The invention relates, generally, to automated methods and system
for detection of chromosomal abnormalities and automated image
analysis in cells for the detection of cancer and dysplasias, more
particularly, for the detection of cancers and dysplasias in
cervical cells. U.S. Application 61/082,346 filed Jul. 21, 2008 is
incorporated herewith in its entirety.
BACKGROUND
Cervical cancer is one of the most common and deadly cancers among
women worldwide. If detected early, cervical cancer and precursor
lesions can be treated effectively. A Pap test is the primary
screen for cervical cancer and uses morphological analysis to
identify suspicious cells. However, a single cytologic examination
is relatively insensitive, poorly reproducible and frequently
yields equivocal results. Approximately 6% of Papanicolaou (Pap)
tests of 50 million performed annually in the United States are
diagnosed as atypical squamous cells of undetermined significance
(ASCUS) and require follow-up testing, and approximately 5% of
ASCUS patients have undetected cancer. Current guidelines for
patients include follow-up Pap testing, testing for human papilloma
virus (HPV) and/or colposcopy.
Current methods for Pap test also include liquid based cytological
sampling and preparation of a monolayer of cells for analysis which
have the additional benefit of use of the samples for HPV
screening.
In addition, approximately 3% of Pap tests of the 50 million
performed annually in the United States are diagnosed with
low-grade squamous intraepithelial lesions (LSIL). Current
guidelines for these patients recommend additional monitoring
and/or colposcopy. Clinical studies show the majority of these
patients are HPV+. There is significant risk for an ASCUS/HPV+ or
LSIL patient to progress to more severe cervical disease and
require surgical treatment within the two years following the
initial test. The identification of these patients that will
progress is impossible based on morphology and HPV infection.
Infection with HPV is associated with cervical cancer and many
patients are tested for HPV after an ASCUS Pap test result or
cotest for Pap/HPV in women whose cytology test is normal but are
HPV positive. All HPV positive women are at risk for disease. The
strength of sensitive HPV testing is that it provides extremely
high negative predictive value; women who test negative are at low
risk for developing cervical cancer. However, the positive
predictive value of HPV testing is limited since only a small
fraction of HPV positive early lesions progress to high-grade
dysplasia and cancer. Thus, HPV detection, even in combination with
cytomorphological evaluation, is a test with poor specificity.
Comparative genomic hybridization (CGH) is a molecular-cytogenetic
method for the analysis of copy number changes (gains/losses) in
the DNA content of a given subject's DNA and often performed on
tumor cells, including cervical cancer. FISH methods can be used
with CGH, array CGH, ELISA, or flow cytometry.
The implementation of cervical cancer screening programs has
greatly reduced disease incidence and mortality in industrialized
countries. However, a single cytological evaluation remains
relatively insensitive, hence the need for frequent follow-up
investigations. This is attributable to sampling or interpretation
errors, and to the fact that some early lesions may not have
acquired recognizable phenotypic alterations. Invasive cervical
carcinomas develop through increasing stages of cervical dysplasia,
to cervical intraepithelial neoplasias, categorized as CIN1, CIN2,
CIN3, and to carcinoma in situ (CIS). While CIN3/CIS are considered
bonafide precancerous lesions, current guidelines indicate surgical
treatment for all CIN2 or more severe lesions. Only about 15% of
all grades dysplastic lesions follow this path of progression. Pap
and HPV tests are indirect methods for determining the presence of
cervical dysplasia or cancer.
Manual methods are presently known to aid in the microscopic
analysis of samples for determining increases or decreases in
chromosomal copy number. By way of example, not limitation, nucleic
acid probes can be labeled and directed to specific chromosomal
structures; such probes are distinguishably labeled, with labels
which fluoresce with different colors. Chromosomal structure may be
elucidated, identified and analyzed using typical techniques of
microscopic detection.
Fluorescent in situ hybridization (FISH) allows for visualization
of genetic material in individual cells. FISH is particularly
versatile because it can be performed on cells that can be actively
or not actively dividing. FISH can be used in a variety of ways,
including the use of locus specific probes to visualize a small
portion of a gene, where the FISH probes only bind to the parts of
the chromosomes to which they have a high degree of sequence
similarity. To visualize the chromosomal region of interest, the
FISH probe must be made to hybridize specifically to a target
sequence, the probes can then be tagged directly with fluorophores
or with targets for antibodies or with biotin.
Specifically, FISH involves the precise annealing of a single
stranded fluorescently labeled DNA probe to complementary target
sequences. The hybridization of the probe with the cellular DNA
site is visible by direct detection using fluorescence microscopy.
After the probes are made, an interphase or metaphase preparation
of the chromosomes is made and is firmly attached to a substrate
such as glass. After contacting the labeled probe with the slides
comprising the prepared cells, the sample is washed to remove any
probes not hybridized. The slide is then scanned via microscopy
after DAPI counterstaining to image the chromosomal regions of
interest.
Typical microscopic automation can provide for efficient and
expedient biological sample analysis. Automatic microscopy can
include, but is not limited to, robotic microscopic systems,
automatic operation, automated slide scanning, automated stage,
automated slide cassettes and handling systems, and computer
software systems to facilitate detection and analysis of
fluorescent signals.
Presently there are no non-manual i.e. automated, methods for the
automatic microscopic analysis of chromosomal abnormalities present
in cervical cells to provide for direct methods of determining the
presence of cervical dysplasia and cancer. Therefore, there remains
a continuing unmet need for automated microscopic methods for
detecting chromosomal abnormalities for the diagnosis of cervical
disease.
In yet another example of probes, ProVysion Multi-color Probe Set
manufactured by Abbott Molecular is designed to detect and quantify
chromosome 8, the lipoprotein lipase (LPL) gene located at 8p22,
and the C-MYC gene located at the 8q24 region. Gain of 8q24 and
8p21-22 (LPL) and loss of heterozygosity are two genetic
alterations that have been observed in abnormal samples. The
ProVysion Multi-color Probe Set consists of three probes with three
separate fluorophore labels. The multicolor probe set design is
said to permit simultaneous analysis of the three genomic markers
within a single cell, CEP.RTM. 8 probe labeled with Spectrum Aqua,
LSI LPL labeled with Spectrum Orange, and LSI C-MYC labeled with
Spectrum Green. The CEP 8 alpha satellite DNA probe hybridizes to
the centromere region of chromosome 8 (8p11.1-q11.1) and provides a
mechanism for the identification of copy number of chromosome 8.
The manufacturer asserts that in a normal cell hybridized with the
ProVysion Multi-color Probe Set, the expected pattern is the two
orange, two green and two aqua (2O2G2A) signal pattern, while in an
abnormal cell, combinations of copies of the three probe signals
may be observed. The test kit indicates that copy numbers of more
or less than two of any probe indicates chromosome or gene gain or
loss, respectively. Less than two copies of the LSI LPL or multiple
copies of the LSI C-MYC Probe relative to CEP 8 copy number
indicates loss of the LPL region and gain of the C-MYC region,
respectively, relative to the chromosome 8 copy number.
U.S. Patent Publication Nos. 2004/028107 and 2005/0026190 to Vysis,
Inc. assert methods of using probes and probe sets for the
detection of high grade dysplasia and carcinoma in cervical cells.
The methods entail hybridizing one or more chromosomal probes to a
biological sample and detecting the hybridization pattern of the
chromosomal probes to determine whether the subject has high grade
dysplasia or carcinoma. The methods encompass the use of a set of
one or more probes demonstrating a vector value of about 60 or less
wherein the vector value is calculated by
Vector=[(100-specificity).sup.2+(100-sensitivity).sup.2].sup.1/2.
The chromosomal probes may comprise probes for specific loci, such
as 8q24, 3q26, Xp22, and CEP 15, or probes, for example,
substantially complementary to full coding sequence for each of
HPV-16, HPV-18, HPV-30, HPV-45, HPV-51, and HPV-58. The biological
sample screened may be pre-screened for the presence of a cell
cycle protein, such as p16 or Cyclin E, or a cell proliferation
marker, such as protein Ki67 or PCNA.
U.S. Patent Publication 2006/0063194 to Abbott Molecular also
discloses probe sets and methods of using probes and probe sets for
the detection of cancer, particularly lung cancer. Locus specific
probes and chromosome enumeration probes are used in conjunction,
and the hybridization pattern of the same used to determine whether
the subject has lung cancer. Chromosomal compositions are
specified, for example, a probe set for determining lung cancer may
comprise a 5p15 locus specific probe, a 8q24 locus specific probe,
a chromosome 6 enumeration probe and a 7p12 locus specific
probe.
SUMMARY
One aspect of the present invention provides for an automated
microscopic method for detecting chromosomal abnormalities, in a
patient sample, in order to determine presence of cervical cell
disease, including cervical cancer or cervical dysplasia
comprising: contacting the patient sample which comprises cervical
cells on a slide with at least two distinguishably labeled probes
directed to a portion of a chromosomal region of the cervical
cells; hybridizing a target nucleic acid region on chromosome 5p,
and more specifically 5p15 in the cervical cells with a first said
labeled nucleic acid probe; hybridizing a target nucleic acid
region on the centromere of chromosome 7 (CEN7), as an aneuploidy
probe, with a second said labeled nucleic acid probe; and
automatically scanning the sample with a microscope to detect the
formation of the hybridization complexes on chromosome 5p and on
CEN7 and to generate an image after sufficient time and conditions
to permit hybridization; automatically analyzing the image to
characterize the chromosomal profile in the cells to generate a
diagnosis; and automatically reporting the diagnosis to a user. In
some aspects, the aneuploidy probe may also be centromere of
chromosome 3 (CEN3).
One aspect of the present invention provides for an automated
microscopic method for detecting chromosomal abnormalities, in a
patient sample, in order to determine presence of cervical cell
disease, including cervical cancer or cervical dysplasia
comprising: contacting the patient sample which comprises cervical
cells on a slide with at least two distinguishably labeled probes
directed to a portion of a chromosomal region of the cervical
cells; hybridizing a target nucleic acid region on chromosome 3q,
and more specifically 3q26 in the cervical cells with a first said
labeled nucleic acid probe; hybridizing a target nucleic acid
region on the centromere of chromosome 7 (CEN7), as an aneuploidy
probe, with a second said labeled nucleic acid probe; and
automatically scanning the sample with a microscope to detect the
formation of the hybridization complexes on chromosome 3q and on
CEN7 and to generate an image after sufficient time and conditions
to permit hybridization; automatically analyzing the image to
characterize the chromosomal profile in the cells to generate a
diagnosis; and automatically reporting the diagnosis to a user. In
some aspects, the aneuploidy probe may also be centromere of
chromosome 3 (CEN3).
In another aspect of the present invention, the first probe can be
directed to a portion of the chromosome region 3q, and more
specifically 3q26. In yet another aspect of the invention, a first
nucleic acid probe targeted to a region of 5p, a second probe
targeted to a region of 3q, and an aneuploidy probe to CEN7, or in
the alternative CEN3, is provided. Each being differentially
labeled such that the probes fluoresce with different colors and
can each be uniquely identified after hybridization with a sample
and subsequent imaging.
As 3q and 5p are specific to carcinogenesis, the aneuploidy probe
permits us to measure carcinogenic processes in general. Also,
genetic alterations have been identified in the early development
of cervical cancer that can predict the patient's risk of disease
progression. These aberrations include gross changes in DNA content
(e.g. ploidy) and the amplification of both a portion of chromosome
3, specifically locus 3q26, that includes a gene TERC that encodes
a subunit of the telomerase protein and a portion of chromosome 5,
specifically 5p15, that includes a gene, TERT, that encodes another
subunit of the telomerase protein, both of which are linked to cell
immortality. Studies have demonstrated multicolor fluorescent DNA
probes can detect abnormalities in both ploidy, and 3q and 5p copy
number by fluorescence in situ hybridization (FISH) with greater
sensitivity and specificity than other methods.
In yet another aspect, the aneuploidy probes, such as CEN7
described herein, can also be a locus specific probe on an arm near
the centromere of chromosome 7 such as probes to 7p12.
Another aspect of the invention provides for a method of screening
for the presence and/or extent of a pathology in a subject, the
pathology characterized by an abnormal chromosomal component in a
cell of the subject, comprising the steps of: contacting a
biological sample comprising cell nuclei from the subject with, one
or more distinguishably labeled probes directed to at least one
chromosomal sequence that characterizes the abnormality under
conditions that promote hybridization of the one or more probes to
the at least one sequence, automatically obtaining a representation
of the one or more distinguishable labels hybridized to the
chromosomal sequences, automatically analyzing the distribution and
intensity of binding of the one or more labels in the
representation to determine the presence and/or extent of an
abnormal chromosomal component; and automatically reporting results
of the analysis; wherein the steps are carried out without
intervention by a human.
Yet another aspect of the invention provides for method of
screening for an abnormality related to a cancer, a high grade
hyperplasia or a high grade dysplasia in a subject, comprising the
steps of: obtaining a biological sample comprising nuclei from the
subject; contacting the biological sample with a first probe
bearing a first detectable label directed to a chromosomal sequence
related to the abnormality under conditions that promote
hybridization of the probes to targeted chromosomal loci;
contacting the sample under the hybridizing conditions with at
least one of a detectably labeled reference probe directed to a
chromosomal locus known not to be abnormal and further contacting
the sample with a nuclear reference stain; automatically finding
areas in the sample having the reference stain and imaging the
labels bound to the chromosomal sequences; automatically analyzing
the label image for the distribution and intensity of hybridized
labels; and automatically reporting results of the analysis;
wherein the steps are performed without intervention by a
human.
These and other aspects of some exemplary embodiments will be
better appreciated and understood when considered in conjunction
with the following description and the accompanying drawings. It
should be understood, however, that the following descriptions,
while indicating preferred embodiments and numerous specific
details thereof, are given by way of illustration and not of
limitation. Many changes and modifications may be made within the
scope of the embodiments without departing from the spirit
thereof.
DESCRIPTION OF THE DRAWINGS
FIG. 1 is a flow diagram of an automated method for diagnosing
cervical cell disease.
FIG. 2 is a flow diagram of another embodiment of the method of the
invention.
FIGS. 3 A, B and C illustrate the stages of cervical cancer
progression as represented by amplification of 3q26 chromosomal
copy number gain.
FIG. 4 illustrates the stages of cervical cancer progression as
represented by amplification of 3q26 and 5p15 chromosomal copy
number gain.
FIG. 5 is an illustration of negative results from a liquid-based
cytology patient sample testing for abnormality in 3q alone.
Interpretation: Evaluation of this specimen has revealed a normal
copy number of the TERC gene. No amplification of the gene at 3q26
was detected and evaluation of the chromosome 7 centromere
indicates a normal diploid cell. This does not rule out other
abnormalities occurring at genetic loci other than those listed
above. Detailed results of the analysis are summarized in the table
above, along with a representative image of cells with normal copy
of TERC. Materials and Methods: Analysis for the Human Telomerase
gene (TERC) was performed using Fluorescent In-Situ Hybridization
(FISH) with a probe specific for the TERC gene on chromosome region
3q26. In addition, a probe specific for chromosome 7 centromere was
applied to assess DNA ploidy. Cervical cells were fixed to a slide
and hybridized with these fluorescent probes. Analysis of the
hybridization was performed to assess the presence of normal and
abnormal cells.
FIG. 6 is an illustration of positive results from a liquid-based
cytology patient sample testing for abnormalities in 3q alone.
Interpretation: Evaluation of this specimen has revealed an
abnormal copy number of the TERC gene. Detailed results of the
analysis are summarized in the table above, along with a
representative image of cells with abnormal copy of TERC. Materials
and Methods: Analysis for the Human Telomerase gene (TERC) was
performed using Fluorescent In-Situ Hybridization (FISH) with a
probe specific for the TERC gene on chromosome region 3q26. In
addition, a probe specific for chromosome 7 centromere was applied
to assess DNA ploidy. Cervical cells were fixed to a slide and
hybridized with these fluorescent probes. Analysis of the
hybridization was performed to assess the presence of normal and
abnormal cells.
FIG. 7 is an illustration of positive results from a liquid-based
cytology patient sample testing for abnormalities in 3q and 5p.
Interpretation: Evaluation of this specimen has revealed an
abnormal copy number of the chromosomal regions 3q26 and 5p15. Gain
of chromosomal region 3q26 has been shown to be an early indicator
of cervical dysplasia, while an increase in copy number at 5p15 is
more often associated with more advanced stages of cervical
carcinoma. Detailed results of the analysis are summarized in the
table above, along with a representative image of cells with
abnormal copy of region 3q26 and/or 5p15. Materials and Methods:
Analysis for the gene specific loci at chromosomal regions 3q26 and
5p15 was performed using Fluorescent In-Situ Hybridization (FISH)
with a probe specific for the TERC gene on chromosome region 3q26
and the Cri du Chat locus at 5p15. In addition, a probe specific
for chromosome 7 centromere was applied to assess DNA ploidy.
Cervical cells were fixed to a slide and hybridized with these
fluorescent probes. Analysis of the hybridization was performed to
assess the presence of normal and abnormal cells.
FIG. 8 is an illustration of negative results from a liquid-based
cytology patient sample testing for abnormalities in 3q and 5p.
Interpretation: Evaluation of this specimen has revealed a normal
copy number of chromosomal regions 3q26 and 5p15. In the number of
cells analyzed, amplification of loci at 3q26 and 5p15 was not
detected and evaluation of the chromosome 7 centromere indicates a
normal diploid specimen. This does not rule out other abnormalities
occurring at sites other than those listed above. Detailed results
of the analysis are summarized in the table above, along with a
representative image of cell(s) with normal copy of loci 3q26
and/or 5p15. Materials and Methods: Analysis for the gene specific
loci at chromosomal regions 3q26 and 5p15 was performed using
Fluorescent In-Situ Hybridization (FISH) with a probe specific for
the TERC gene on chromosome region 3q26 and the Cri du Chat locus
at 5p15. In addition, a probe specific for chromosome 7 centromere
was applied to assess DNA ploidy. Cervical cells were fixed to a
slide and hybridized with these fluorescent probes. Analysis of the
hybridization was performed to assess the presence of normal and
abnormal cells.
FIGS. 9 A and B is an illustration of negative results from a
patient's tissue biopsy sample testing for abnormalities in 3q
alone. FIG. 8A is from a CIN1 tissue biopsy. FIG. 8B is from a CIN2
biopsy.
FIGS. 10 A, B, and C is an illustration of positive results from a
patient's tissue biopsy sample testing for abnormalities in 3q
alone. FIG. 9A is from a CIN1 tissue biopsy. FIG. 9B is from a CIN2
tissue biopsy. FIG. 9C is from a CIN3 tissue biopsy.
FIG. 11 is an illustration of results from a patient's tissue
biopsy sample testing for abnormalities in 3q and 5p.
FIG. 12 is an illustration of a liquid-based cytology patient
sample testing for abnormalities in 3q alone with tetraploid
cells.
FIG. 13 is an illustration of a liquid-based cytology patient
sample testing for abnormalities in 3q and 5p tetraploid cells.
FIGS. 14 A and B illustrates genomic organization of chromosomal
locus 3q26, including the gene position of TERC, among other genes,
and BAC clones suitable for the production of labeled DNA
probes.
FIGS. 15 A, B, C, and D illustrate a listing of genes that can be
targeted by specific probes of the invention to measure chromosomal
abnormalities in cervical cells.
DETAILED DESCRIPTION
The present invention is based on the identification of gain in
copy number of chromosomal regions associated with cancer, and in
particular cervical cancer.
The present invention provides for automated microscopic method for
detecting chromosomal abnormalities in order to determine presence
of cervical cell disease, including cervical cancer or cervical
dysplasia, in a patient sample comprising: contacting the patient
sample comprising cervical cells on a slide with at least two
distinguishably labeled probes directed to a portion of a
chromosomal region of the cervical cells; hybridizing a target
nucleic acid region on chromosome 5p in the cervical cells with a
first the labeled nucleic acid probe; hybridizing a target nucleic
acid region on centromere of chromosome 7 (CEN7) with a second said
labeled nucleic acid probe; and automatically scanning the sample
with a microscope to detect the formation of the hybridization
complexes on chromosome 5p and on CEN7 and to generate an image
after sufficient time and conditions to permit hybridization;
automatically analyzing the image to characterize the chromosomal
profile in the cells to generate a diagnosis; and automatically
reporting the diagnosis to a user.
The present invention provides for automated microscopic method for
detecting chromosomal abnormalities in order to determine presence
of cervical cell disease, including cervical cancer or cervical
dysplasia, in a patient sample comprising: contacting the patient
sample comprising cervical cells on a slide with at least two
distinguishably labeled probes directed to a portion of a
chromosomal region of the cervical cells; hybridizing a target
nucleic acid region on chromosome 3q in the cervical cells with a
first the labeled nucleic acid probe; hybridizing a target nucleic
acid region on centromere of chromosome 7 (CEN7) with a second said
labeled nucleic acid probe; and automatically scanning the sample
with a microscope to detect the formation of the hybridization
complexes on chromosome 3q and on CEN7 and to generate an image
after sufficient time and conditions to permit hybridization;
automatically analyzing the image to characterize the chromosomal
profile in the cells to generate a diagnosis; and automatically
reporting the diagnosis to a user.
As 3q and 5p are specific to carcinogenesis, the aneuploidy probe
permits us to measure carcinogenic processes in general. These
aberrations include gross changes in DNA content (e.g. ploidy) and
the amplification of both a portion of chromosome 3, specifically
locus 3q26, that includes a gene TERC that encodes a subunit of the
telomerase protein and a portion of chromosome 5, specifically
5p15, that includes a gene, TERT, that encodes another subunit of
the telomerase protein, both of which are linked to cell
immortality. Studies have demonstrated multicolor fluorescent DNA
probes can detect abnormalities in both ploidy, and 3q and 5p copy
number by fluorescence in situ hybridization (FISH) with greater
sensitivity and specificity than other methods. Therefore, in
another embodiment of the invention, a first nucleic acid probe
targeted to a region of 5p, a second probe targeted to a region of
3q, and an aneuploidy probe to CEN7, or in the alternative CEN3, is
provided. Each being differentially labeled such that the probes
fluoresce with different colors and can each be uniquely identified
after hybridization with a sample and subsequent imaging.
In yet another embodiment, the aneuploidy probes, such as CENT
described herein, can also be a locus specific probe on an arm near
the centromere of chromosome 7 such as probes to 7p12.
It is yet another embodiment of the invention to provide for an
automated microscope and system to perform each of the steps of the
method disclosed herein. It is an embodiment of the invention
whereby each of the step is carried out without manual
intervention. It is also an embodiment of the invention, for the
microscope to read a patient identified, e.g. barcode, on the slide
for entry into a database prior to scanning so that the results of
the method can be indexed according to each patient identifier.
In another embodiment, provided for is a method of screening for
the presence and/or extent of a pathology in a subject, the
pathology characterized by an abnormal chromosomal component in a
cell of the subject, comprising the steps of: contacting a
biological sample comprising cell nuclei from the subject with, one
or more distinguishably labeled probes directed to at least one
chromosomal sequence that characterizes the abnormality under
conditions that promote hybridization of the one or more probes to
the at least one sequence, automatically obtaining a representation
of the one or more distinguishable labels hybridized to the
chromosomal sequences, automatically analyzing the distribution and
intensity of binding of the one or more labels in the
representation to determine the presence and/or extent of an
abnormal chromosomal component; and automatically reporting results
of the analysis; wherein the steps are carried out without
intervention by a human. A description of such methods can be found
in US Patent Publication Numbers US 20090250629, US 20090208965, US
20080182253, and US 20080213769, each of which is hereby
incorporated by reference in their entirety.
Yet another embodiment is a method of screening for an abnormality
related to a cancer, a high grade hyperplasia or a high grade
dysplasia in a subject, comprising the steps of: obtaining a
biological sample comprising nuclei from the subject; contacting
the biological sample with a first probe bearing a first detectable
label directed to a chromosomal sequence related to the abnormality
under conditions that promote hybridization of the probes to
targeted chromosomal loci; contacting the sample under the
hybridizing conditions with at least one of a detectably labeled
reference probe directed to a chromosomal locus known not to be
abnormal and further contacting the sample with a nuclear reference
stain; automatically finding areas in the sample having the
reference stain and imaging the labels bound to the chromosomal
sequences; automatically analyzing the label image for the
distribution and intensity of hybridized labels; and automatically
reporting results of the analysis; wherein the steps are performed
without intervention by a human. A description of such methods can
be found in US Patent Publication Numbers US 20090250629, US
20090208965, US 20080182253, and US 20080213769, each of which is
hereby incorporated by reference in their entirety.
Because cancer is a genetic disease, and genetic aberrations can be
observed in diseased cells, the present method provides for
automatic and efficient diagnosis of cervical disease by measuring
such genetic abnormalities. The aberrations can be observed
cytologically, by measuring genetic aberrations either as increase,
hyperplasia, or decrease, hypoplasia, in copy number of gene
regions collectively dysplasia. Also, certain chromosomal copy
number differences are evident in cancer cells such that
measurement of aneuploidy can be a diagnostic indicator of disease
state in the cell, whether or not it can be observed cytologically.
The methods discussed herein can directly identify abnormalities in
the DNA of cervical cells using fluorescently labeled probes that
bind to the aberrant regions in the chromosome. When greater than,
or less than, the expected number of signals are observed, a cell
sample can be diagnosed as diseased and cervical dysplasia can be
diagnosed before it can be observed cytologically. Patients with
these abnormalities can have a poor prognosis and can be at high
risk to develop more advanced cervical disease. The methods
disclosed herein can be performed subsequent to or in lieu of
normal Pap, i.e. negative for intrathelial legions or malignancy
(NILM), NILM/HPV+, HPV+, ASCUS/HPV+ or LSIL Pap tests, among other
abnormal results from cytology testing, in order to provide more
specific information about a patient's risk of disease
progression.
As used herein, "copy number gain" or "amplification" means any
chromosomal copy number greater than normal in a human pattern.
As used herein, "cervical cell disease" means any of the following:
cervical carcinogenesis, Human Papilloma Virus (HPV) positive,
Atypical Squamous Cells of Undetermined Significance (ASC-US),
Low-grade Squamous Intraepithelial Lesion (LSIL), Atypical Squamous
Cells, HSIL (ASC-H), Atypical Glandular Cells of Undetermined
Significance (AGUS), High-grade Squamous Intraepithelial Lesion
(HSIL), cervical dysplasia, pre-cancer, pre-malignant legion,
cervical cancer, cervical adenocarcinoma, cervical squamous cell
carcinoma, cervical intraepithelial neoplasia 1 (CIN1), cervical
intraepithelial neoplasia (CIN2), cervical intraepithelial
neoplasia 3 (CIN3), carcinoma in situ, invasive cervical carcinoma,
and cytological or genetic abnormality of the cell and can be
interchangeably used with cervical cancer or cervical dysplasia.
Also, "disease," "cell disease," or "disease" as used herein
includes but is not limited to any cytological or genetic
abnormality of the cell.
"Human Papilloma Virus positive," "HPV+," or "HPV positive" as used
herein means any HPV subtype identified by standard diagnostic HPV
testing kits, such as those available from Qiagen of Maryland and
Hologic of Massachusetts, including HPV subtypes 16, 18. The
definition also includes the presence of HPV mRNA or the expression
of HPV proteins, including E6, E7.
The present method provides direct identification of genetic
abnormalities in morphologically normal cells and abnormal cells,
as well as prognostic information about disease progression. In
certain embodiments of the method, it may be used with squamous and
glandular cervical cells or identify morphological characteristics
that may be indicators of disease.
Copy Number Gains in Cervical Cancer
An increase in 3q copy number, in addition to integration of human
papilloma virus (HPV) into the host genome, have been associated
with the progression of CIN2 or CIN3 to cervical carcinoma and both
appear to be important associated events in the progression of
cervical dysplasia to invasive cancer. Hopman et al. J. Pathol.
2006 December: 210(4): 412-9. Higher staged tumors or those with
lymph node metastasis had more chromosomal imbalances including
gains of 3q; 1q; 8q; and losses of 11q; 3q; 6q and 2q. Gains of
3q11-q22 and 3q26-qter were more prevalent with lymph node
metastasis. Huang, K. F. et al., J. Formos Med Assoc. 2007
November; 106(11): 894-902.
3q gains seen in invasive cervical carcinomas, specifically gain in
the human telomerase gene (TERC), have been used in the development
of FISH probe sets as a diagnostic tool in the detection of TERC
gains in Pap smears. It has been suggested that TERC gains could
predict progression from CIN1/CIN2 to CIN3 and invasive carcinoma.
Heselmeyer-Haddad et al. Am Journal of Pathology 2005;
166:1229-1238.
5p is also a frequently observed structurally changed chromosome in
carcinomas. Atkin, N. B 1997 Elsevier; 95: 33-39. Arias-Pulido, H.
et al. 2002 Mol. Cancer; 1:3. Huang F. Y., et al. 2005 Cancer Gen.
and Cyto., 157: 46-47. Macville M., et al. 1999 Cancer Res.;
59:141-50. Heselmeyer K. et al. 1997 Genes Chromosomes Cancer; 19:
233-40. Rao P. H. et al. 2004 BMC Cancer; 4:5. 5p gains are
observed during progression to advanced stage carcinomas, and
frequently involve whole arm amplifications. Heselmeyer K. et al.
1997 Genes Chromosomes Cancer; 19: 233-40.
Using carcinoma cell lines that showed 5p amplification, a minimal
region of alteration at 5p13.33 has been defined, which encodes the
human telomerase reverse transcriptase (hTERT) gene. Lockwood, W.
et al. Int. J. Cancer 2006; 120: 436-443. Finally, an HPV
integration site has also been mapped to 5p11-15. Lockwood, W. et
al. Int. J. Cancer 2006; 120: 436-443. Telomerase activation is a
component of cancer cell immortality Takuma, Y. et al. 2004 Journal
of Gastroenterology and Hepatology; 19: 1300-1304. Takahashi S. et
al. 2000 Eur. Jour. Of Cancer, 36: 496-502. Toshikuni, N. et al.
2000 Br. J. Cancer; 82; 833-837. hTERT has been identified as the
catalytic subunit of telomerase. Takahashi S. et al. 2000 Eur.
Jour. Of Cancer, 36: 496-502.
hTERT expression has been observed in several cancer cell lines,
including cervical carcinomas, with certain cancer cell lines
showing that hTERT expression is high in cancerous lesions but not
non-cancerous tissues. Takahashi S. et al. 2000 Eur. Jour. Of
Cancer, 36: 496-502. Although this differential expression was
found in hepatocarcinomas rather than cervical carcinomas, the
results suggest that hTERT expression occurred at an early stage of
hepatocarcinogenesis. Takahashi S. et al. 2000 Eur. Jour. Of
Cancer, 36: 496-502. Further, 5p hTERT gene amplification is
closely correlated with increased hTERT mRNA expression in cervical
cancers with HPV infection. Zhang A. et al. 2000 Cancer Res.; 60:
6230-6235. Zhang A. et al. 2002 Genes Chromosomes Cancer; 34:
269-75. 5p has been consistently identified as a chromosome that
undergoes structural changes during various stages of
carcinogenesis. The structural changes also appear to consistently
affect the TERT gene encoded on 5p.
5p gain has been observed in invasive cervical carcinoma. Scotto,
et al., Molecular Cancer 2008. When observed in samples in addition
to observations of gain in 3q, specifically 3q26, it can be an
indicator of increased progression of disease state from cervical
dysplasia to invasive cervical carcinoma. Using a genomic probe for
a region on 3q, specifically chromosome band 3q26, in combination
with at least one aneuploidy probe, (eg. CEP3, CEN7), and a genomic
probe for a region on 5p, especially 5p15, the copy number
increases precede malignant conversion of cervical intraepithelial
neoplasms to invasive carcinoma, and further accompany the
transition from ASCUS or CIN1 to CIN2 or CIN3 and from CIN2 or CIN3
to carcinoma in situ to invasive cervical carcinoma. Moreover, the
identification of gain in both 3q and 5p indicates an expedited
transition from ASCUS or CIN1 to CIN2 or CIN3 and from CIN2 or CIN3
to carcinoma in situ to invasive cervical carcinoma.
The present methods provide for automated identification of
possible cervical cell disease by comparing the copy number
increase of the target chromosomes, for example, 5p, 3q or both
together, as compared to normal. As used herein, "normal" means
chromosomal diploidy in mammalian cells except when cells that are
normally diploid are tetraphase and in the cell cycle and
tetraploidy is observed. Chromosomal diploidy is measured by
targeting a centromeric region, e.g. CEN3 or CEN7, with a probe.
The automated methods can be used as a diagnostic and prognostic
marker for cervical dysplasia in patients with ploidy abnormalities
and/or increased 3q and/or increased 5p copy numbers. Such methods
for diagnosing cervical cell disease are fully disclosed in related
applications U.S. Provisional Ser. No. 61/082,346; U.S. application
Ser. No. 12/506,985; U.S. Provisional Ser. No. 61/227,270; U.S.
Provisional Ser. No. 61/249,720; and U.S. application Ser. No.
10/540,311, each of which is incorporated by reference herein in
its entirety, including any referenced cited therein.
The automated microscopy methods disclosed herein may further
comprise, in addition to the use of distinguishably labeled probes
for the detection of genetic amplification in chromosome 3q and 5p,
the use of probes, distinguishably labeled for hybridization to
regions of: 1q; 20q; 12q; 19q; 11 q; 6q; 17p; 7; 8q, which is
detected in late stage dysplasia; 9q; 16q; 2q; 9p; 10q; 18p and any
combination thereof.
According to specific embodiments of the invention, amplification
in the 3q26 locus and 5p15 locus band can be detected. In yet
further specific embodiments of the invention, in addition to
detection the 3q26 and 5p15 loci, amplification in the following
chromosomal loci can be detected: 1q21-31; 20q12; 12q13-24; 19q13;
11q21; 7q11-22; 8q24 which is detected in late stage dysplasia;
9q33-34; 16q23; 2q32; 9p22; 10q21-24; 18p11 and any combination
thereof.
The method can further comprise determining that the genomic
amplification of chromosome 3q and/or chromosome 5p is not present
in the sample. When compared to a state where aneuploidy is found
and cervical cell disease is identified, the loss of chromosomal
amplification can be indicative of regression of cell disease and
possibly regression of disease. The method can include compiling
individual patient data in a database whereby the results are
studied for each sample. Subsequent test from the same patient are
compared to prior recorded results to determine progression on
regression of disease.
Generally, the automated method according to claim 1, 2 or 3,
wherein said method analyzes a shift in patient condition from low
grade to high grade cervical dysplasia or cancer or from high grade
to low grade dysplasia; predisposition for or likelihood of
developing a cervical cell disease; maintenance or regression of a
patient condition; confirms or verifies the successful treatment of
a cervical cell disease or monitors for recurrence and
effectiveness of treatment; or quantifies the extent and/or
severity of a cervical cell disease. The methods may also be used
for assessing and monitoring late stage dysplasia comprising
detecting genomic amplification in chromosomes 3q, as well as 5p
and 8q, more specifically 8q24. Gain of 8q copy number and/or gain
in 5p copy number in combination with gain in 3q, can indicate
malignant conversion of cervical intraepithelial neoplasms to
invasive carcinoma, and further accompany the transition from
ASCUS/CIN1 to CIN2/CIN3 and from CIN2/CIN3 to carcinoma in situ to
invasive cervical carcinoma and even to metastasis. Moreover, the
identification of gain in both 3q and 5p indicates an expedited
transition from ASCUS/CIN1 to CIN2/CIN3 and from CIN2/CIN3 to
carcinoma in situ to invasive cervical carcinoma. Further, the
method disclosed herein can be used to identify metastatic
potential and metastasis of the disease.
In another embodiment, HPV infections may be involved in
development of anal cancers because of the similar biology between
cervical and anal carcinogenesis, including similar cell types and
viral initiation, genomic abnormalities and copy number changes
occur at 3q and 5p among other loci. Because anal cancer in
patients approaches the rates of cervical cancer in women screening
for HPV and/or bio-markers related to cervical cancer as disclosed
herein can screen for anal cancer. It is therefore a further
embodiment of the present invention to analyze anal cell specimens
for 3q and 5p among the other chromosomal copy number changes to
determine whether a patient may have anal disease. Therefore, these
methods could be used on anal specimens and provide valuable
clinical information regarding anal carcinogenesis.
Probes
A number of methods can be used to identify probes which hybridize
specifically to the specific loci exemplified herein. For instance,
probes can be generated by the random selection of clones from a
chromosome specific library, and then mapped by digital imaging
microscopy. This procedure is described in U.S. Pat. No. 5,472,842,
herein incorporated by reference in its entirety. Various libraries
spanning entire chromosomes are also available commercially from
for instance Illumina Inc. Probes that hybridize specific
chromosomal loci are available commercially from Abbot Molecular,
Inc. (Des Plaines, Ill.).
Briefly, a genomic or chromosome specific DNA is digested with
restriction enzymes or mechanically sheared to give DNA sequences
of at least about 20 kb and more preferably about 40 kb to 300 kb.
Techniques of partial sequence digestion are well known in the art.
See, for example Perbal, A Practical Guide to Molecular Cloning,
2nd Ed., Wiley N.Y. (1998). The resulting sequences are ligated
with a vector and introduced into the appropriate host. Exemplary
vectors suitable for this purpose include cosmids, yeast artificial
chromosomes (YACs), bacterial artificial chromosomes (BACs) and P1
phage. Various libraries spanning entire chromosomes are also
available commercially.
Once a probe library is constructed, a subset of the probes is
physically mapped on the selected chromosome. FISH and digital
image analysis can be used to localize clones along the desired
chromosome. Briefly, the clones are mapped by FISH to metaphase
spreads from normal cells using e.g., FITC as the fluorophore. The
chromosomes may be counterstained by a stain which stains DNA
irrespective of base composition (e.g., DAPI or propidium iodide),
to define the outlining of the chromosome. The stained metaphases
are imaged in a fluorescence microscope with a polychromatic
beam-splitter to avoid color-dependant image shifts. The different
color images are acquired with a CCD camera and the digitized
images are stored in a computer. A computer program is then used to
calculate the chromosome axis, project the two (for single copy
sequences) FITC signals perpendicularly onto this axis, and
calculate the average fractional length from a defined position,
typically the p-telomere. This approach is described, for instance,
in U.S. Pat. No. 5,472,842, herein incorporated by reference in its
entirety.
Sequence information of the genes identified here permits the
design of highly specific hybridization probes or amplification
primers suitable for detection of target sequences from these
genes. As noted above, the complete sequence of these genes or
chromosomal regions can be identified by means known to those of
skill in the art. For instance, oligonucleotide probes chosen to be
complementary to a selected subsequence within the gene can be
used. Alternatively, sequences or subsequences may be amplified by
a variety of DNA amplification techniques (for example via
polymerase chain reaction, ligase chain reaction, transcription
amplification, etc.) prior to detection using a probe.
Amplification of DNA increases sensitivity of the assay by
providing more copies of possible target subsequences. In addition,
by using labeled primers in the amplification process, the DNA
sequences may be labeled as they are amplified.
Other probes used can be made by isolating DNA from BAC clones and
labeling DNA. Chromosomal probes are typically about 50 to about
1.times.10.sup.5 nucleotides in length. Longer probes typically
comprise smaller fragments of about 100 to about 500 nucleotides in
length. Probes that hybridize with centromeric DNA and
locus-specific DNA are available commercially, for example, from
Abbott Molecular, Inc. (Downers Grove, Ill.), Life Technologies,
Inc. of California or Veridex of New Jersey. Alternatively, probes
can be made non-commercially from chromosomal or genomic DNA
through standard techniques. For example, sources of DNA that can
be used include genomic DNA, cloned DNA sequences, somatic cell
hybrids that contain one, or a part of one, human chromosome along
with the normal chromosome complement of the host, and chromosomes
purified by flow cytometry or microdissection. The region of
interest can be isolated through cloning, or by site-specific
amplification via the polymerase chain reaction (PCR). See, for
example, Nath and Johnson, Biotechnic Histochem., 1998, 73(1):6-22,
Wheeless et al., Cytometry 1994, 17:319-326, and U.S. Pat. No.
5,491,224.
In a preferred embodiment, probes of the present invention may be
directed to at least a portion of TERC gene, at band 3q26.2, and
TERT, TRIP13, at 5p15.3 or Cri du Chat locus at 5p15.2.
Specifically, a probe to TERC at region 3q26 can be used labeled
with spectrum gold and also a probe for 5p15 labeled with spectrum
green. Such probes are commercially available from Abbot Molecular
(Des Plaines, Ill.). However, the probes of the invention can
include any gene on the 3q26 and 5p15 including, but not limited
to, those referenced herein including those listed in FIG. 15 and
any combination or portion of the genes on 3q26 or 5p15. In other
embodiments, the target nucleic acid target comprises PIK3CA, PRCKI
or GLUT2.
In a specific embodiment, the detectable marker of the probe can
emit a fluorescent signal or the probe may be chromogenic. The
probes are hybridized using FISH as described herein. The present
invention provides for an automated fluorescence microscopy
platform that can be used to find out where the fluorescent probe
binds to the chromosome. In instances where additional genetic
material is required for testing, the genome may be amplified or
detected by Polymerase Chain Reaction (PCR).
In another embodiment, the automated microscopy method as used
herein comprises a procedure of performing FISH on liquid cytology
specimens, such as SUREPATH.RTM. or THINPREP.RTM., as well as
tissue samples, or others as are known in the art, specimens for
successful hybridization of DNA probes of the automated method
described herein. SUREPATH.RTM. is available from Becton-Dickinson
of Sparks, Md. THINPREP.RTM. is available from Hologic Laboratories
of Bedford, Mass.
It is yet another aspect of the invention to use antibodies to
separate squamous and glandular cells out of liquid-based cytology
specimens prior to detecting genetic amplification in sample cells.
The separation of cell types can improve detection of both squamous
and glandular cancers and improve detection of cervical carcinomas
which are rarely detected through traditional Pap testing but show
3q26 amplification, 5p15 amplification, or both. Glandular cells
are lower in the tissue and present in the endocervical canal and
abnormalities are likely to be missed in typical testing. If
abnormalities are observed the likelihood of detecting and
diagnosing cervical adenucarcinomas based on the analysis of
glandular cells is increased as compared to testing of squamous
cells or a mixed sample of squamous and glandular cells.
As used herein "label" or "labels" is any composition, e.g. probe,
detectable by spectroscopic, photochemical, biochemical,
immunochemical, or chemical means including but not limited to
fluorescent dyes (e.g. fluorescein, rhodamine, Texas Red, etc.,
enzymes, electron dense reagents, magnetic labels, and the like).
Labels which are not directly detected but are detected through the
use of indirect label include biotin and dioxigenin as well as
haptens and proteins for which labeled antisera or monoclonal
antibodies are available. Methods of labeling nucleic acids and
probes are well known to those of skill in the art. Preferred
labels are those that are suitable for use in in situ
hybridization. The nucleic acid probes may be detectably labeled
prior to hybridization. Alternatively, a detectable label which
binds to the hybridization product may be used. Such detectable
labels include any material having a detectable physical or
chemical property and are well developed in the field of
immunoassays.
Typically, it is desirable to use multiple color, in a preferred
embodiment three-color FISH methods for detecting chromosomal
abnormalities in which three probes are utilized, each labeled by a
different fluorescent dye. In the preferred embodiment, two test
probes that hybridizes to the regions of interest are labeled with
two different dyes and an aneuploidy probe that hybridizes to a
different region is labeled with a third dye. More than three
probes can be used so long as each is labeled with a unique dye. A
nucleic acid probe that hybridizes to a stable region of the
chromosome of interest such as the centromere, is preferred as an
aneuploidy probe so that differences between efficiency of
hybridization from sample to sample can be determined.
In a preferred embodiment comprising differentially labeled probes,
the labeled probe panel may consist at least of a three-color,
three-probe mixture of DNA probe sequences homologous to specific
regions on chromosomes 3, 5 and 7; and/or other chromosome regions
disclosed herein.
It is an embodiment of the system and method to be used in
conjunction with specimens in liquid suspension, i.e. thin layer
cytology specimen or thin layer suspension, that can be placed onto
a microscope slide in an even, monolayer of cells, this includes
liquid-base cytology specimens such as THINPREP.RTM. and
SUREPATH.RTM. plus any fine-needle aspirate (FNA), sputum, or
swab-based collection.
Cells recovered and isolated from specimens or samples collected
from patients can be fixed on slides. Specimens can be retrieved
using various techniques known in the art. In one embodiment
specimens can be retrieved from THINPREP.RTM. and/or SUREPATH.RTM.
samples. SUREPATH.RTM. is a Pap test used for the screening of
cervical cancer. The THINPREP.RTM. Pap is also a liquid-based
cytology method. A sample of the cervical cells is rinsed into a
vial instead of a smear onto a slide thus preventing clumping of
cells. The cells are separated in a laboratory to eliminate blood
and mucus and the cells to be studied are then placed on a slide
for studies to detect cancerous cells.
In addition to liquid or tissue sample from Pap, the method may
also comprise analysis of tissue from cervical biopsies, punch
biopsies, surgical procedures including LEEP, hysterectomy, CONE
biopsy, ECC, CONE biopsy. The sample may be prepared from tissue or
cells removed from the cervix, vagina, vulva or vaginal cuff,
uterus, ovary or fallopian tube. The cells may be in metaphase or
interphase.
In yet another embodiment, the nuclei can be isolated from a sample
using methods known to those of skill in the art to/from single
nuclei preparation from tissue samples. Wangsa et al., American
Journal of Pathology, Vol. 175, No. 6, December 2009, incorporated
in its entirety by reference herein.
Hybridization
In an embodiment, the regions disclosed here are identified using
in situ hybridization. Generally, in situ hybridization comprises
the following major steps: (1) fixation of tissue or biological
structure to be analyzed; (2) pre-hybridization treatment of the
biological structure to increase accessibility of target DNA, and
to reduce nonspecific binding; (3) hybridization of the mixture of
nucleic acids to the nucleic acid of the biological sample or
tissue; (4) post-hybridization washes to remove nucleic acid
fragments not bound in the hybridization and (5) detection of the
hybridized nucleic acids. Hybridization protocols for the
applications described herein are described in U.S. Pat. No.
6,277,563, incorporated herein by reference in its entirety.
From samples, the target DNA can be denatured to its single
stranded form and subsequently allowed to hybridize with the probes
of the method. Following hybridization, the unbound probe is
removed by a series of washes, and the nuclei are counterstained
with DAPI (4, 6 diamidino-2-phenylindole), a DNA-specific stain.
Hybridization of the DNA probes can be viewed using a fluorescence
microscope equipped with appropriate excitation and emission
filters allowing visualization of the aqua and gold fluorescent
signals. Enumeration of CEN 7,5p15 and 3q26 signals is conducted by
microscopic examination of the nuclei.
The clinical test disclosed herein can use several biomarkers in
combination for the early detection of cervical cancer and is
important because current morphology based screening and detection
methods have significant limitations. Identification of 3q26 and
5p15, among others, amplification and other cytogenetic
abnormalities can more precisely and accurately identify patients
at risk for developing cervical cancer and help them receive
earlier treatment.
Image Analysis
The present invention provides for automatic image analysis and
scoring of the methods disclosed via an automated microscope. The
automated hybridization of the probe with the cellular DNA site is
visible by direct detection using fluorescence microscopy as
described herein. In a preferred embodiment, the probe panel
consists of a 3-color, three-probe mixture of DNA probe sequences
homologous to specific regions on chromosomes 3, 5, and 7. The
probe mixture consists of a locus specific probe for chromosome
3q26, 5p15, and centromere of chromosome 7 (CEN7).
It is an embodiment of the present invention to provide for
automated image analysis of the signal from the FISH probe.
Microscopes can allow for automated capture of digital images of
the field of view within the specimen/slide on the microscopy
stage. Such manufacturers include Carl Zeiss, Leica, Nikon and
Olympus. Also, the method provides for software platforms for
automated image analysis such as microscope-software systems
developed by such entities Applied Spectral Imaging of California,
as Ikonisys of Connecticut, Metasystems of Massachusetts and
Germany, Bioimagene of California, and Bioview of Massachusetts and
Israel, among others. Such automated systems may apply to viewing
3q chromosomes alone or in combination with 5p abnormalities in the
patient sample.
Cells recovered from specimens can be fixed on slides. The target
DNA is denatured to its single stranded form and subsequently
allowed to hybridize with the probes. Following hybridization, the
unbound probe can be removed by a series of washes, and the nuclei
are counterstained with DAPI (4,6 diamidino-2-phenylindole), a
DNA-specific stain. Hybridization of the probes can be viewed using
a fluorescence microscope equipped with appropriate excitation and
emission filters allowing visualization of the three fluorescent
signals. Enumeration of CEN7, 5p15 and 3q26 signals is conducted by
automated microscopic examination of the nuclei.
The probe set and DAPI counterstain can be viewed on an automated
epi-fluorescence microscope equipped with a 100-watt mercury lamp
equipped with the following filters: DAPI, Spectrum Aqua
(chromosome 7 centromere), Spectrum Green (locus on 5p15), and
Spectrum Orange (locus on 3q26) or other labels and probes as are
known in the art and disclosed herein. The automated microscopy
with DAPI filter and a magnification of 10.times., and 2.times.,
4.times. or 5.times. at the initial scan or subsequent passes, can
automatically scan sample area of patient slide to determine cell
quantity and quality. Analysis can begin in the upper left quadrant
of the target area and scan fields with 63.times. oil, 40.times. or
20.times. or 63.times. or 100.times. or greater, objective from
left to right and top to bottom without re-scanning the same areas.
The system can count a total of about 1000 cells. The slides can be
automatically located and unloaded onto the microscopy stage. After
scanning, the computer software system automatically analyses the
image and provides for automatic scoring of the cell counts and
delivery of a report to a user.
Determination of chromosomal copy number in at least 800 cells, and
preferably 1000 cells, is a preferred sampling of each clinical
specimen. Less than 800 cells or more than 1000 cells can also be
utilized in this system. The method and system overcome sampling
variations and limitations of slide production methodology. The
methods and system are consistent with methods recommended by
professional medical organizations (ACMG) to determine the
threshold between a specimen with and without chromosomal copy
number changes. Wolf, D. J. et al. (2007) Period Guidelines for
Fluorescence In Situ Hybridization Testing.
The automated method and system provides for at least 90% accuracy
for positive specimens and identifies a patient with an increased
risk of disease progression. The method and system can further
provide for greater than 95% accuracy.
In situ hybridization is a technique that allows the visualization
of specific nucleic acid sequences within a cellular preparation.
Traditionally the visualization of probe signals has been performed
manually by highly-trained personnel. Microscopes that can be used
in the present method include, but are not limited to, those
manufactured by Carl Zeiss, Leica, Nikon, and Olympus, which allow
the user to capture digital images of the field of view within the
specimen/slide on the microscopy stage. Software systems of the
invention automatically acquire images from automatically loaded
specimens/slide for high through put analysis. The automated
systems include both a microscopy platform and the automated
imaging software.
The type and source of the specimen to be analyzed directly impacts
the analysis process and methodology. Each tissue type has its own
biology and structure plus each cancer develops differently with
different factors affecting the rate of carcinogenesis. In order to
account for variation in cell biology, morphology and structure,
the method can distinguish between epithelial and other cells and
structures to avoid unwanted artifacts in the image. The software
system of the invention can account for these different factors.
Morphology can be automatically imaged where cells morphogenically
suspicious for malignancy can be further analyzed for morphological
abnormalities including, but not limited to, pyknosis, large
nuclear size, irregular nuclear shape, and patchy DAPI staining
Therefore, the system can begin with cells that appear
morphologically abnormal before counting normal cells. If few
morphologically abnormal cells are present, cells which are the
largest or have the largest detectable nuclei are scanned and
analyzed. Overlapping cells that cannot be distinguished are not
counted.
Further, another embodiment of the invention comprising identifying
clusters or clumps of cells for morphological signals in
abnormalities where said clusters can be indicative of more
advanced cervical disease such as CIN2 or more severe disease.
Scoring and Analysis
The automated system then analyzes the image to automatically
enumerate each probe signal within the DAPI-stained region and
records the copy number of each probe identified. The software
system continues its automated scoring of cells and chromosomal
copy number within each cell until it obtains results of at least
800 cells in some embodiments 100, 200 or more cells. Once the 800
cell threshold is reached, the software can categorize each cell
imaged and counted into a category based upon the copy number of
each chromosome identified. By way of example, not limitation,
normal cell with two copies of each probe 3q26, 5p15, and CEN7
would be placed into a 2, 2, 2 category. Abnormal cells would be
identified by their probe signal patterns. For instance, a cell
with two copies of the CEN7 probe, 5 copies of the 3q26 probe and 3
copies of the 5p15 probe can be placed in the 2, 5, 3 category.
Once all of the imaged cells are categorized, the specimen can be
evaluated relative to the positive/negative disease threshold. All
cells identified as abnormal by the automated imaging system can be
reviewed and verified manually by trained personnel before test
results are communicated to a physician. The method and system
further provides for automated verification. Specific cell
threshold numbers can vary by specimen type and collection method.
In addition, the software can be adapted to reflect biological,
e.g. cell shape, cell size, DNA content of the nucleus, proximity
of cells to each other, cell type, etc., or disease related
differences, e.g. number of loci with abnormal number, the number
of abnormalities at a locus within a single cell, relationship of
an abnormality to survival or treatment response. This method and
system can be used on a representative sampling of area covered by
cells on the slide instead of the entire area, typically this is
performed by imaging multiple fields of view or a path based on
cellular density until the minimum imaged cell threshold is
met.
In one embodiment, all cells identified as abnormal by the
automated system are communicated electronically via methods known
in the art to a physician or other user. Only a subset of the
rank-ordered abnormal cells can be reviewed relative to the
positive/negative test threshold as long as the clinical and
disease significance is known for the subset. Typically the subset
is the most abnormal about 25 or about 50 cells within the
specimens, but other subsets can be identified and utilized
depending on the specimen source, collection method, and
disease.
Yet another embodiment can be used in conjunction with tissue-based
specimens such as those from a biopsy or surgical procedure. In
addition, this system and method can use a companion slide that is
stained with hematoxylin and eosin stain (H&E) that comes from
the same tissue-based specimen. This automated method screens the
entire area covered by tissue-based specimen on the FISH prepared
slide and utilizes the DAPI-stain to identify cellular nuclei. The
system then enumerates each probe signal within the DAPI-stained
region and records the copy number of each probe identified. The
software system continues its automated scoring of cells and
chromosomal copy number within each cell until the entire
tissue-based specimen has been reviewed. The software then
evaluates a sub-section of the slides that contains at least 25
nuclei as identified by the DAPI stain. The selection on the
sub-section location is guided based upon disease indicators on the
companion H&E slide. Typically, at least two sub-sections are
selected for each specimen. The software then categorizes each cell
imaged and counted into a category based upon the copy number of
each chromosome identified.
In yet another embodiment, the system and method captures an image
used alternatively for scoring by (1) identifying the image sample
number and recording the image used (2) visualizing the signal
colors separately (3) analyzing and recording the signal patterns
for individual nuclei, selecting the appropriate nuclei based on
the criteria described in preceding paragraph and (4) recording the
signal numbers.
The software can automatically score data by: scanning the sample;
recording and analyzing the image; calculating the number of any
one of the signals, e.g. 3q, 5p, or CENT, or other targeted regions
as disclosed herein, and dividing by the total number of nuclei
scored; recording that number in a Scoring Database. A result
greater than 2 is recorded and reported as amplified for any given
probe and is noted as abnormal and possible cervical cell disease.
Images can be named by the specimen number and slide number and
saved.
It is a preferred embodiment of the invention disclosed herein
whereas all steps may be performed without human intervention.
EXAMPLES
Example 1
Methods and Specimen Selection for Automated Analysis and
Scoring
A total of twenty-seven (27) specimens were identified within the
tested population that had negative results for cytology, HPV and
two-color method testing. In addition, these 27 specimens all had
1000 total cells counted in order to minimize variation within the
analysis. The triple negative specimens were considered disease
negative and, therefore, any abnormal cells identified by FISH
would be considered `false positives.` Once the number of `false
positive` cells is determined for the 27 specimens, the described
BETAINV calculation can be used to determine the threshold between
normal and abnormal specimens.
The results of FISH testing for the 27 triple negative specimens
were reviewed to identify the total number of `false positive`
cells per specimen at each chromosomal loci. A specimen had 5 cells
identified by FISH to be abnormal across both loci, 3q26 and 5p15,
the greatest number of observed `false positive` cells per
specimen. This observation was used within the BETAINV calculation
to determine the threshold between normal and abnormal specimens
95% confidence. The total number of cells entered into the equation
was 1000. The BETAINV calculation returned a threshold value of
0.0104 (1.0%) or 10 cells out of 1000 counted cells. The threshold
was also determined with 99% confidence and was equal to 0.0129
(1.3%) or 13 cells out of 1000.
In addition, the results of FISH testing for the 27 triple negative
specimens were reviewed to identify the total number of `false
positive` cells per specimen at chromosomal locus 3q26 (TERC). The
specimen had 3 cells identified by FISH to be abnormal 5p15, the
greatest number of observed `false positive` cells per specimen.
This observation was used within the BETAINV calculation to
determine the threshold between normal and abnormal specimens 95%
confidence. The total number of cells entered into the equation was
1000 from ND10107B. The BETAINV calculation returned a threshold
value of 0.0077 (0.7%) or 7 cells out of 1000 counted cells. The
threshold was also determined with 99% confidence and was equal to
0.0104 (0.0104%) or 10 cells out of 1000.
In addition, the results of FISH testing for the 27 triple negative
specimens were reviewed to identify the total number of `false
positive` cells per specimen at chromosomal locus 5p15 (Cri du
Chat) and 3q26. The specimen had 3 cells identified by FISH to be
abnormal 3q26, the greatest number of observed `false positive`
cells per specimen. This observation was used within the BETAINV
calculation to determine the threshold between normal and abnormal
specimens 95% confidence. The total number of cells entered into
the equation was 1000. The BETAINV calculation returned a threshold
value of 0.0104 (1.0%) or 10 cells out of 1000 counted cells. The
threshold was also determined with 99% confidence and was equal to
0.0129 (1.3%) or 13 cells out of 1000.
Using the ACMG guidelines, the threshold between normal and
abnormal specimens was determined for the automated analysis of the
present method. With 95% confidence, the threshold was determined
to be 10 cells out of 1000 cells or 1.0% abnormal cells per
specimen. Therefore, any specimen with 1.0% or greater percentage
of abnormal cells by FISH is abnormal and identifies a patient with
a higher risk of progression. In these cases, a POSITIVE diagnostic
test result can be issued. When a specimen is found to have less
than 1.0% abnormal cells per specimen, a NEGATIVE diagnostic test
report can be issued.
Example 2
FISH was performed on previously prepared thin layer, liquid-based
cytology samples (THINPREP.RTM., Cytyc, Marlborough, Mass.). Slides
were made from THINPREP.RTM. vials and then subject to a
pretreatment protocol that includes protease digestion,
formaldehyde fixation, washing, and dehydration. Hybridization was
performed using a two-color multi-target interphase FISH probe kit
(Abbot Molecular). The kit included directly labeled probes to
CEN7-aqua and to the locus of 3q26 (3q-orange) and to the locus of
5p15 (5p-green). The cells were analyzed using fluorescence
microscopy.
Samples had a minimum of 800 cells for analysis. Positive tests
showed aneuploidy and (1) gains of either 3q copy number or 5p copy
number in 1.0% or more of the analyzed cells; (2) gains of only 3q
copy number of 0.9% or more of the analyzed cells; or (3) gains of
only 5p copy number in 0.7% or more of the analyzed cells. Negative
tests showed normal ploidy and (1) less than 1.0% of analyzed cells
with an increase in both 3q copy number and 5p copy number; (2)
gains of only 3q copy number in less than 0.9% of the analyzed
cells; or (3) gains of only 5p copy number in less than 0.7% of the
analyzed cells. Samples with ploidy abnormalities and/or increased
3q copy number were determined to have a poor prognosis and risk to
develop more advanced cervical disease.
Results can be a diagnostic and prognostic marker for cervical
dysplasia. Samples with ploidy abnormalities and/or increased 3q
copy number and 5p copy number were determined to have a poor
prognosis and risk to develop more advanced cervical disease.
Example 3
Evaluation of FIG. 7 this specimen has revealed an abnormal copy
number of the TERC gene on 3q26 and the Cri du Chat locus 5p15. Of
1000 cells analyzed, 986 cells were normal while 8 were found
abnormal for extra copies of TERC (3q), 2 were found abnormal for
extra copies of Cri du Chat (5p), and 4 were found abnormal for
extra copies of TERC and Cri du Chat (3q and 5p) for an abnormal
cell percentage of 1.40%.
Example 4
Evaluation of FIG. 8 this specimen has revealed a normal copy
number of the TERC gene on 3q26 and the Cri du Chat locus on 5p15.
Of 1000 cells analyzed, 998 cells were normal while 2 were found
abnormal for extra copies of TERC (3q), 0 were found abnormal for
extra copies of Cri du Chat (5p), and 0 were found abnormal for
extra copies of TERC and Cri du Chat (3q and 5p) for an abnormal
cell percentage of 0.20%.
Example 5
Analysis for the Human Telomerase gene (TERC), 3q26, was performed
using Fluorescent In-Situ Hybridization (FISH) with a probe
specific for the TERC gene on chromosome region 3q26. In addition,
a probe specific for CENT was applied to assess DNA ploidy.
Cervical cells were fixed to a slide and hybridized with these
fluorescent probes. Analysis of the hybridization was performed to
assess the presence of normal and abnormal cells.
Example 6
Evaluation of FIG. 6 this specimen has revealed an abnormal copy
number of the TERC gene. Along with a representative image of cells
with an abnormal copy number of TERC. Of 1030 cells analyzed, 1012
cells were normal while 18 were found abnormal for an abnormal cell
percentage of 1.85%.
Example 7
Evaluation of FIG. 5 revealed a normal copy number of the TERC
gene. No amplification of the gene at 3q26 was detected and
evaluation of the chromosome 7 centromere indicates a normal
diploid cell. This does not rule out other abnormalities occurring
at sites other than those listed above. Along with a representative
image of cells with a normal copy of TERC. Of 800 cells analyzed,
799 cells were normal while 1 was found abnormal for an abnormal
cell percentage of 0.1%.
Example 8
Tissue Fish
FISH was performed on 4-micron thick tissue sections cut from
formalin fixed paraffin-embedded (FFPE) tissue specimens. (FIGS. 8
and 9) Slides were subject to a pretreatment protocol that includes
protease digestion, washing, and dehydration. Hybridization was
performed using a two-color FISH probe set containing directly
labeled probes to CEN7-aqua and to the locus of 3q26 (3q-orange)
(Probes obtained from Abbott Molecular). The sections were
counterstained with DAPI and the cells were analyzed using
fluorescence microscopy.
A minimum of fifty cells per sample were analyzed for ploidy
status. Samples were judged aneuploid if the ratio of 3q26 probe
signal to nuclei within the selected cells was 2.0 or greater.
Samples were judged to have normal ploidy if the ratio of 3q26
probe signals to nuclei within the selected cells was less than 2.
Patients with ploidy abnormalities and/or increased 3q copy number
were determined at risk for a poor prognosis and are at high risk
to develop more advanced cervical disease.
Results can be a diagnostic and prognostic marker for cervical
dysplasia. Samples with ploidy abnormalities and/or increased 3q
copy number were determined at risk for poor prognosis and to
develop more advanced cervical disease.
Example 9
FISH was performed on 4-micron thick tissue sections cut from
formalin fixed paraffin-embedded (FFPE) tissue specimens. (FIG. 10)
Slides were subject to a pretreatment protocol that includes
protease digestion, washing, and dehydration. Hybridization was
performed using a three-color FISH probe set. The set included
directly labeled probes to CEN7-aqua and to the locus of 3q26
(3q-orange) and to the locus of 5p15 (5p-green) (probes obtained
from Abbott Molecular). The sections were counterstained with DAPI
and the cells were analyzed using fluorescence microscopy. A
minimum of fifty cells per sample were analyzed for ploidy
status.
Samples were judged to have aneuploidy if (1) the ratio of 3q26
probe signal to nuclei within the selected cells was about 2.0 or
greater; (2) the ratio of 5p15 probe signal to nuclei within the
selected cells was about 2.0 or greater; (3) the ratio of 3q26 and
5p15 probe signals to nuclei within the selected cells was about
2.0 or greater. Samples were judged to have normal ploidy if the
ratio of 3q26 or 5p15 or both 3q26 & 5p15 probe signals to
nuclei within the selected cells was less than 2. Samples with
ploidy abnormalities and/or increased 3q copy number were
determined at risk for poor prognosis and at risk to develop more
advanced cervical disease.
The foregoing description of some specific embodiments provides
sufficient information that others can, by applying current
knowledge, readily modify or adapt for various applications such
specific embodiments without departing from the generic concept,
and, therefore, such adaptations and modifications should and are
intended to be comprehended within the meaning and range of
equivalents of the disclosed embodiments. It is to be understood
that the phraseology or terminology employed herein is for the
purpose of description and not of limitation. In the drawings and
the description, there have been disclosed exemplary embodiments
and, although specific terms may have been employed, they are
unless otherwise stated used in a generic and descriptive sense
only and not for purposes of limitation, the scope of the claims
therefore not being so limited. Moreover, one skilled in the art
will appreciate that certain steps of the methods discussed herein
may be sequenced in alternative order or steps may be combined.
Therefore, it is intended that the appended claims not be limited
to the particular embodiment disclosed herein.
Each of the applications and patents cited in this text, as well as
each document or reference cited in each of the applications and
patents (including during the prosecution of each issued patent;
"application cited documents"), and each of the PCT and foreign
applications or patents corresponding to and/or claiming priority
from any of these applications and patents, and each of the
documents cited or referenced in each of the application cited
documents, are hereby expressly incorporated herein by reference.
More generally, documents or references are cited in this text,
either in a Reference List before the claims; or in the text
itself; and, each of these documents or references ("herein-cited
references"), as well as each document or reference cited in each
of the herein-cited references (including any manufacturer's
specifications, instructions, etc.), is hereby expressly
incorporated herein by reference.
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